Locomotor exercise and circadian rhythms in mammals

The timing of locomotor activity is a major rhythmic output of the mammalian circadian system but both spontaneous and induced exercise can also themselves impact on circadian rhythms across levels of organisation and in multiple tissues, including both the brain and periphery. This review briefly summarises historical research on the influences of locomotor activity and its correlates on circadian function and then discusses recent advances in this area. Locomotor exercise can acutely alter circadian phase, both of overt behavioural rhythms and in peripheral tissues, and can stably entrain rhythms in behaviour driven by the hypothalamic master circadian pacemaker in the suprachiasmatic nucleus (SCN). Locomotor activity potently and acutely suppresses both electrical activity and clock gene expression in the master circadian pacemaker of the hypothalamus, though mechanistically how this is achieved is still unclear, as are the effects of locomotor activity in diurnal species, including humans. © 2018 Elsevier Lt

Delayed Cryptochrome Degradation Asymmetrically Alters the Daily Rhythm in Suprachiasmatic Clock Neuron Excitability.

Suprachiasmatic nuclei (SCN) neurons contain an intracellular molecular circadian clock and the Cryptochromes (CRY1/2), key transcriptional repressors of this molecular apparatus, are subject to post-translational modification through ubiquitination and targeting for proteosomal degradation by the ubiquitin E3 ligase complex. Loss-of-function point mutations in a component of this ligase complex, Fbxl3, delay CRY1/2 degradation, reduce circadian rhythm strength, and lengthen the circadian period by ∼2.5 h. The molecular clock drives circadian changes in the membrane properties of SCN neurons, but it is unclear how alterations in CRY1/2 stability affect SCN neurophysiology. Here we use male and femaleAfterhoursmice which carry the circadian period lengthening loss-of-functionFbxl3Afhmutation and perform patch-clamp recordings from SCN brain slices across the projected day/night cycle. We find that the daily rhythm in membrane excitability in the ventral SCN (vSCN) was enhanced in amplitude and delayed in timing inFbxl3Afh/Afhmice. At night, vSCN cells fromFbxl3Afh/Afhmice were more hyperpolarized, receiving more GABAergic input than theirFbxl3+/+counterparts. Unexpectedly, the progression to daytime hyperexcited states was slowed byAfhmutation, whereas the decline to hypoexcited states was accelerated. In long-term bioluminescence recordings, GABAAreceptor blockade desynchronized theFbxl3+/+but not theFbxl3Afh/AfhvSCN neuronal network. Further, a neurochemical mimic of the light input pathway evoked larger shifts in molecular clock rhythms inFbxl3Afh/Afhcompared withFbxl3+/+SCN slices. These results reveal unanticipated consequences of delaying CRY degradation, indicating that theAfhmutation prolongs nighttime hyperpolarized states of vSCN cells through increased GABAergic synaptic transmission.SIGNIFICANCE STATEMENTThe intracellular molecular clock drives changes in SCN neuronal excitability, but it is unclear how mutations affecting post-translational modification of molecular clock proteins influence the temporal expression of SCN neuronal state or intercellular communication within the SCN network. Here we show for the first time, that a mutation that prolongs the stability of key components of the intracellular clock, the cryptochrome proteins, unexpectedly increases in the expression of hypoexcited neuronal state in the ventral SCN at night and enhances hyperpolarization of ventral SCN neurons at this time. This is accompanied by increased GABAergic signaling and by enhanced responsiveness to a neurochemical mimic of the light input pathway to the SCN. Therefore, post-translational modification shapes SCN neuronal state and network properties

A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus

Background: In mammals, the synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior. However, the dominance of this clock has been challenged by the observations that metabolic duress can over-ride SCN controlled rhythms, and that clock genes are expressed in many brain areas, including those implicated in the regulation of appetite and feeding. The recent development of mice in which clock gene/protein activity is reported by bioluminescent constructs (luciferase or luc) now enables us to track molecular oscillations in numerous tissues ex vivo. Consequently we determined both clock activities and responsiveness to metabolic perturbations of cells and tissues within the mediobasal hypothalamus (MBH), a site pivotal for optimal internal homeostatic regulation. Results: Here we demonstrate endogenous circadian rhythms of PER2::LUC expression in discrete subdivisions of the arcuate (Arc) and dorsomedial nuclei (DMH). Rhythms resolved to single cells did not maintain long-term synchrony with one-another, leading to a damping of oscillations at both cell and tissue levels. Complementary electrophysiology recordings revealed rhythms in neuronal activity in the Arc and DMH. Further, PER2::LUC rhythms were detected in the ependymal layer of the third ventricle and in the median eminence/pars tuberalis (ME/PT). A high-fat diet had no effect on the molecular oscillations in the MBH, whereas food deprivation resulted in an altered phase in the ME/PT. Conclusion: Our results provide the first single cell resolution of endogenous circadian rhythms in clock gene expression in any intact tissue outside the SCN, reveal the cellular basis for tissue level damping in extra-SCN oscillators and demonstrate that an oscillator in the ME/PT is responsive to changes in metabolism

Cellular mechano-environment regulates the mammary circadian clock

Circadian clocks drive B24 h rhythms in tissue physiology. They rely on transcriptional/ translational feedback loops driven by interacting networks of clock complexes. However, little is known about how cell-intrinsic circadian clocks sense and respond to their microenvironment. Here, we reveal that the breast epithelial clock is regulated by the mechano-chemical stiffness of the cellular microenvironment in primary cell culture. Moreover, the mammary clock is controlled by the periductal extracellular matrix in vivo, which contributes to a dampened circadian rhythm during ageing. Mechanistically, the tension sensing cell-matrix adhesion molecule, vinculin, and the Rho/ROCK pathway, which transduces signals provided by extracellular stiffness into cells, regulate the activity of the core circadian clock complex. We also show that genetic perturbation, or age-associated disruption of self-sustained clocks, compromises the self-renewal capacity of mammary epithelia. Thus, circadian clocks are mechano-sensitive, providing a potential mechanism to explain how ageing influences their amplitude and function

Acute Suppressive and Long-Term Phase Modulation Actions of Orexin on the Mammalian Circadian Clock

Circadian and homeostatic neural circuits organize the temporal architecture of physiology and behavior, but knowledge of their interactions is imperfect. For example, neurons containing the neuropeptide orexin homeostatically control arousal and appetitive states, while neurons in the suprachiasmatic nuclei (SCN) function as the brain's master circadian clock. The SCN regulates orexin neurons so that they are much more active during the circadian night than the circadian day, but it is unclear whether the orexin neurons reciprocally regulate the SCN clock. Here we show both orexinergic innervation and expression of genes encoding orexin receptors (OX1 and OX2) in the mouse SCN, with OX1 being upregulated at dusk. Remarkably, we find through in vitro physiological recordings that orexin predominantly suppresses mouse SCN Period1 (Per1)-EGFP-expressing clock cells. The mechanisms underpinning these suppressions vary across the circadian cycle, from presynaptic modulation of inhibitory GABAergic signaling during the day to directly activating leak K+ currents at night. Orexin also augments the SCN clock-resetting effects of neuropeptide Y (NPY), another neurochemical correlate of arousal, and potentiates NPY's inhibition of SCN Per1-EGFP cells. These results build on emerging literature that challenge the widely held view that orexin signaling is exclusively excitatory and suggest new mechanisms for avoiding conflicts between circadian clock signals and homeostatic cues in the brain

Circadian Disruptions in the Myshkin Mouse Model of Mania are Independent of Deficits in Suprachiasmatic Molecular Clock Function

Background Alterations in environmental light and intrinsic circadian function have strong associations with mood disorders. The neural origins underpinning these changes remain unclear, although genetic deficits in the molecular clock regularly render mice with altered mood-associated phenotypes. Methods A detailed circadian and light-associated behavioral characterization of the Na+/K+-ATPase (NKA) α3 Myshkin (Myk/+) mouse model of mania was performed. NKA α3 does not reside within the core circadian molecular clockwork, but Myk/+ mice exhibit concomitant disruption in circadian rhythms and mood. The neural basis of this phenotype was investigated through molecular and electrophysiological dissection of the master circadian pacemaker, the suprachiasmatic nuclei (SCN). Light input and glutamatergic signalling to the SCN were concomitantly assessed through behavioral assays and calcium imaging. Results In vivo assays revealed several circadian abnormalities including lengthened period and instability of behavioral rhythms, and elevated metabolic rate. Grossly aberrant responses to light included accentuated resetting, accelerated re-entrainment and an absence of locomotor suppression. Bioluminescent recording of circadian clock protein (PER2) output from ex vivo SCN revealed no deficits in Myk/+ molecular clock function. Optic-nerve crush rescued the circadian period of Myk/+ behavior, highlighting that afferent inputs are critical upstream mediators. Electrophysiological and calcium imaging SCN recordings demonstrated changes in response to glutamatergic stimulation as well as electrical output indicative of altered retinal input processing. Conclusions The Myshkin model demonstrates profound circadian and light-responsive behavioral alterations independent of molecular clock disruption. Afferent light-signaling drives behavioral changes and raises new mechanistic implications for circadian disruption in affective disorders

Timed exercise stabilizes behavioral rhythms but not molecular programs in the brain's suprachiasmatic clock

Timed daily access to a running-wheel (scheduled voluntary exercise; SVE) synchronizes rodent circadian rhythms and promotes stable, 24h rhythms in animals with genetically targeted impairment of neuropeptide signaling (Vipr2−/− mice). Here we used RNA-seq and/or qRT-PCR to assess how this neuropeptide signaling impairment as well as SVE shapes molecular programs in the brain clock (suprachiasmatic nuclei; SCN) and peripheral tissues (liver and lung). Compared to Vipr2+/+ animals, the SCN transcriptome of Vipr2−/− mice showed extensive dysregulation which included core clock components, transcription factors, and neurochemicals. Furthermore, although SVE stabilized behavioral rhythms in these animals, the SCN transcriptome remained dysregulated. The molecular programs in the lung and liver of Vipr2−/− mice were partially intact, although their response to SVE differed to that of these peripheral tissues in the Vipr2+/+ mice. These findings highlight that SVE can correct behavioral abnormalities in circadian rhythms without causing large scale alterations to the SCN transcriptome

Timed daily exercise remodels circadian rhythms in mice

Regular exercise is important for physical and mental health. An underexplored and intriguing property of exercise is its actions on the body’s 24 h or circadian rhythms. Molecular clock cells in the brain’s suprachiasmatic nuclei (SCN) use electrical and chemical signals to orchestrate their activity and convey time of day information to the rest of the brain and body. To date, the long-lasting effects of regular physical exercise on SCN clock cell coordination and communication remain unresolved. Utilizing mouse models in which SCN intercellular neuropeptide signaling is impaired as well as those with intact SCN neurochemical signaling, we examined how daily scheduled voluntary exercise (SVE) influenced behavioral rhythms and SCN molecular and neuronal activities. We show that in mice with disrupted neuropeptide signaling, SVE promotes SCN clock cell synchrony and robust 24 h rhythms in behavior. Interestingly, in both intact and neuropeptide signaling deficient animals, SVE reduces SCN neural activity and alters GABAergic signaling. These findings illustrate the potential utility of regular exercise as a long-lasting and effective non-invasive intervention in the elderly or mentally ill where circadian rhythms can be blunted and poorly aligned to the external world

Circadian Oscillators in the Epithalamus

The habenula complex is implicated in a range of cognitive, emotional and reproductive behaviors, and recently this epithalamic structure was suggested to be a component of the brain's circadian system. Circadian timekeeping is driven in cells by the cyclical activity of core clock genes and proteins such as per2/PER2. There are currently no reports of rhythmic clock gene/protein expression in the habenula and therefore the question of whether this structure has an intrinsic molecular clock remains unresolved. Here, using videomicroscopy imaging and photon-counting of a PER2::luciferase (LUC) fusion protein together with multiunit electrophysiological recordings, we tested the endogenous circadian properties of the mouse habenula in vitro. We show that a circadian oscillator is localized primarily to the medial portion of the lateral habenula. Rhythms in PER2:: LUC bioluminescence here are visualized in single cells and oscillations continue in the presence of the sodium channel blocker, tetrodotoxin, indicating that individual cells have intrinsic timekeeping properties. Ependymal cells lining the dorsal third ventricle also express circadian oscillations of PER2. These findings establish that neurons and non-neuronal cells in the epithalamus express rhythms in cellular and molecular activities, indicating a role for circadian oscillators in the temporal regulation of habenula controlled processes and behavior